Direct presentation of inflammation-associated self-antigens by thymic innate-like T cells induces elimination of autoreactive CD8+ thymocytes

Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens. Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system. Here, we demonstrate that CD8+ T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules. In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets. We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8+ thymocytes. Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.

Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens.Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system.Here, we demonstrate that CD8 + T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules.In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets.We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8 + thymocytes.Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.
The generation of a diverse antigen receptor repertoire through V(D) J recombination inevitably produces self-reactive receptors in some developing lymphocytes.To prevent autoimmunity, these self-reactive cells must be inactivated either at the site of development (central tolerance) or in secondary lymphoid organs (peripheral tolerance).Central tolerance requires the presence of antigens at the site of lymphocyte differentiation.In the thymus, several mechanisms ensure the breadth of thymocyte exposure to peripheral antigens.A wide range of tissue-restricted antigens show 'ectopic' expression in medullary thymic epithelial cells (mTECs) [1][2][3] , and some mTECs induce large parts of molecular programs of various peripheral cell types [4][5][6][7] .The thymus also harbors all major subsets of dendritic cells (DCs) that can present both endogenously expressed and TEC-derived antigens 8,9 .Migration of peripheral DCs into the thymus further expands the spectrum of tissue-specific antigens represented in the thymus 8,10 .Finally, another professional antigen-presenting cell (APC) type, thymic B cells, also contributes to the induction of T cell tolerance [11][12][13] .
Self-reactive thymocytes are inactivated by negative selection 14 , eviction of immature cells from the thymus 15 and diversion into lineages with regulatory or innate-like properties 16 .Differentiation of innate-like However, many innate-like T cells found in the thymus, including a large fraction of γδT cells and the majority of invariant natural killer T (iNKT) cells, are not newly developed immature cells but mature tissue-resident lymphocytes 23,24 .Inflammatory mediators secreted by these cells have a broad impact on other components of the thymic microenvironment.For example, type 2 cytokines derived from innate-like cells support the intrathymic generation of innate-like T cells represents the execution of one of the specialized effector programs (for example, type 1 helper T (T H 1) cell-, type 2 helper T (T H 2) cellor interleukin-17 (IL-17)-producing helper T (T H 17) cell-like 17,18 ) following antigen encounter in the thymus.These cells can express genes encoding inflammatory mediators and cytotoxic molecules at steady state [18][19][20] and often home to barrier tissues, where they contribute to the first line of defense against pathogens and regulate tissue homeostasis 21,22 .b, Uniform manifold approximation and projections (UMAPs) highlighting the expression of indicated genes in the mouse thymus cell atlas scRNA-seq dataset; AU, arbitrary units.
https://doi.org/10.1038/s41590-024-01899-6interferon-γ (IFNγ)-producing CD8 + T cells 25,26 , regulate the recruitment of thymic eosinophils 27 , affect TEC function 28 and induce the activation of thymic DCs 29 .Intriguingly, other inflammatory pathways, for example, type 1/type 3 IFN signaling and MyD88-dependent signaling in TECs 30,31 , are also constitutively activated in the thymus.Therefore, the thymus has evolved to harbor a complex constitutively active inflammatory network, and thymus-resident innate-like T cells represent one of its central nodes.The reasons for the activation of numerous inflammatory pathways in the organ where pathogen encounter is unlikely remain unclear.
If autoreactive T cells escape central tolerance and encounter antigen in secondary lymphoid organs in the absence of inflammation, they are inactivated by peripheral tolerance mechanisms 32 .Mature T lymphocytes will only undergo clonal expansion and differentiation into effector cells if they have received co-stimulatory signals that are provided by APCs only in the context of inflammation.The induction of T cell effector programs whose components constitute self-antigens therefore coincides both spatially and temporally with the presence of a pathogen.Breach of tolerance to such antigens would result in a 'fratricide' reaction that would curtail any further immune response involving expression of this molecule.It remains unclear how the immune system discriminates such inflammation-associated self-antigens from antigens originating from the pathogen itself.
In this study, we aimed to investigate the mechanisms of tolerance to T cell-derived inflammation-associated self-antigens.Using model inflammation-associated self-antigens, we demonstrate that CD8 + T cell tolerance to these molecules can be induced in the thymus.The high efficiency of this tolerance was ensured by multiple redundant cellular sources of these antigens in the thymus, which, in addition to TECs, included thymic innate-like T cells and thymic eosinophils.Expression of a self-antigen by minute numbers of thymic innate-like T cells was sufficient to mediate remarkably efficient elimination of autoreactive CD8 + thymocytes that occurred through direct T cell-to-T cell antigen presentation.These results suggest that constitutive expression of inflammation-associated molecules in the thymus may have evolved to ensure the induction of tolerance to this unique class of self-antigens.

Innate-like T cells as libraries of T cell effector antigens
To start unraveling mechanisms of tolerance induction to T cellderived inflammation-associated self-antigens, we analyzed the expression of a selection of genes encoding T cell effector molecules (Il4, Il13, Ifng, Il17a, Il17f and Gzmb) and their upstream regulators (Eomes and Tbx21) in thymic cell subsets.Analysis of bulk transcriptomics data from thymic cell types available in the Immunological Genome (Immgen) database 33,34 (Fig. 1a) and in the integrated single-cell RNA-sequencing (scRNA-seq) mouse thymus atlas 6,35,36 (Fig. 1b) demonstrated that professional thymic APCs, such as TECs and thymic DCs, express no or low levels of many of these molecules.Instead, the highest levels of expression of most of these molecules in the thymus overlapped with the expression of Zbtb16, the gene encoding the transcription factor PLZF that orchestrates effector programs of many innate-like T cell populations [37][38][39][40] (Fig. 1b).Analysis of the Immgen dataset confirmed predominant expression of effector T cell molecules in iNKT and γδT cell subsets (Fig. 1a).This pattern of expression was also conserved in humans as determined by analysis of data from the human scRNA-seq thymus atlas 35 (Extended Data Fig. 1).Thus, in contrast to professional APCs, thymic innate-like T cells express high levels of effector molecules characteristic of all major effector T cell subsets and would represent perfect libraries of T cell effector program antigens.

CD8 + T cell tolerance to a model inflammation self-antigen
To start characterizing tolerance to inflammation-associated self-antigens, we first took advantage of the predominant expression of Il4 by innate-like T cells in the mouse thymus (Fig. 1a,b) and exploited green fluorescent protein (GFP) in Il4-IRES-GFP mice (referred to hereafter as IL-4-GFP mice) 41 as a generic model of antigen expressed by thymic innate-like T cells.Immunization with a major histocompatibility complex (MHC) class I (H2-K d ) GFP 200-208 epitope 42 resulted in a prominent CD8 + T cell response in wild-type (WT) mice, but no response was detected in IL-4-GFP animals (Fig. 2a), indicating a remarkably efficient CD8 + T cell tolerance to GFP in this system.By contrast, immunization with an MHC class II (I-A b ) GFP 81-95 epitope 43 resulted in comparable CD4 + T cell responses in WT and IL-4-GFP mice (Fig. 2b).Thus, expression of IL-4-GFP resulted in strong CD8 + , but not CD4 + , T cell tolerance.
Next, to assess the contribution of hematopoietic and stromal cells to the induction of CD8 + T cell tolerance, we established WT → WT, IL-4-GFP → WT, WT → IL-4-GFP and IL-4-GFP → IL-4-GFP bone marrow (BM) chimeras and immunized them with the GFP 200-208 peptide.A clear expansion of GFP-specific CD8 + T cells was observed only in the WT → WT group, whereas the frequency of tetramer + cells in all other groups was similar to that in unimmunized controls (Fig. 2c).Together, these results reveal efficient induction of CD8 + T cell tolerance to a model inflammation-associated self-antigen, with a redundant role of hematopoietic and radioresistant cells in this process.

Central tolerance to a model inflammation self-antigen
We next sought to assess if tolerance to IL-4-GFP was already established in the thymus.Analysis of IL-4-GFP expression revealed that only 0.5 ± 0.2% (mean ± s.d.) of hematopoietic cells in the thymus were GFP + , of which 76.9 ± 3.3% were innate-like T cells and 17.7 ± 3.4% represented thymic eosinophils (Fig. 3a and Extended Data Fig. 2a,b).GFP expression was also detectable in 0.8 ± 0.2% of mTECs (Extended Data Fig. 2c).Immunofluorescence microscopy revealed that GFP + SiglecF +
We next tested if the expression of IL-4-GFP by hematopoietic and/or stromal cells would result in the inactivation of autoreactive CD8 + T cells already in the thymus.We took advantage of Jedi T cell antigen receptor-αβ ( Jedi-TCRαβ) mice, in which T cells express prerearranged chains of the H2-K d GFP 200-208 -specific TCR from the endogenous Tcra and Tcrb loci 44 .We generated BM chimeras by transferring a mix of Jedi-TCRαβ and WT or Jedi-TCRαβ and IL-4-GFP BM cells into WT or IL-4-GFP recipients and analyzed T cell development in these four groups of BM chimeras.As expected, in the absence of IL-4-GFP, the Jedi thymocyte compartment in WT + Jedi-TCRαβ → WT chimeras included CD4/CD8-double-negative (DN), CD4/CD8-double-positive (DP) and CD8-single-positive (CD8SP) cells (Fig. 3c).Strikingly, expression of IL-4-GFP solely by thymic hematopoietic cells in the IL-4-GFP + Jedi-TCRαβ → WT group or by radioresistant cells in the WT + Jedi-TCRαβ → IL-4-GFP group in both cases resulted in a virtually complete elimination of GFP-specific CD8SP thymocytes (Fig. 3c recipients upregulated PD-1 expression and contained a large fraction of dying cells (Fig. 3c) and cells positive for active caspase-3 (Fig. 3d), indicating that these autoreactive cells were eliminated by negative selection.Strikingly, in IL-4-GFP + Jedi-TCRαβ → WT chimeras, this elimination of autoreactive thymocytes was mediated by as few as 0.1 ± 0.02% of GFP + hematopoietic cells present in the thymi of these mice (Extended Data Fig. 2e).By contrast, WT + Jedi-TCRαβ → IL-4-GFP chimeras contained, on average, 0.01% of residual GFP + hematopoietic cells, and some had virtually no detectable GFP + thymocytes, indicating that, in this setting, induction of tolerance is likely to be mediated solely by thymic stroma (Extended Data Fig. 2e).The disappearance of Jedi CD8 + thymocytes was reflected in the periphery by an overall decrease in Jedi-TCRαβ cells and a near complete loss of GFP-specific conventional CD8 + T cells in the spleen (Fig. 3e).As the TCR in Jedi-TCRαβ mice was expressed prematurely at the DN stages of T cell development (Fig. 3c), we performed a similar analysis with Jedi-TCRβ mice that only carry the TCRβ chain of the Jedi TCR and thus rely on recombination of the endogenous Tcra loci at the DP stage of T cell development.
A small distinct population of Jedi-TCRβ GFP-specific CD8 + T cells was completely eliminated in the presence of antigen (see Supplementary Note 1 and Supplementary Figs. 1 and 2).We conclude that most CD8 + T cells that recognize IL-4-GFP are inactivated already in the thymus, and TECs and hematopoietic cells play redundant roles in the induction of central tolerance to this inflammation-associated self-antigen.

iNKT cells and eosinophils can mediate negative selection
As the thymic GFP + population in IL-4-GFP mice, although dominated by innate-like T lymphocytes, represent a complex mix of cells (Fig. 3a), we next aimed to test if antigen expression solely by innate-like T cells was sufficient to induce tolerance.To this end, we established reaggregate thymus organ cultures (RTOCs; Fig. 4a) with small numbers of Jedi DP thymocytes and iNKT cells sorted from WT or IL-4-GFP thymi.The presence of as few as 0.2% of GFP + iNKT cells in these thymus organoids resulted in a near complete deletion of Jedi cells with few remaining cells acquiring the CD4 -CD8α lo CD8β lo PD-1 hi cell-surface phenotype (Fig. 4b).Thus, antigen expression by minute numbers of innate-like T cells is sufficient to eliminate autoreactive thymocytes in the RTOC system.Of note, this highly efficient induction of negative selection was not a unique property of iNKT cells, as small numbers of conventional ACTB-GFP-transgenic CD4SP thymocytes and thymic eosinophils from IL-4-GFP mice likewise induced elimination of Jedi-TCRαβ DP thymocytes in RTOCs (Extended Data Fig. 3a-c).These results suggest that in addition to professional thymic APCs, such as TECs, negative selection of autoreactive CD8 + thymocytes can be induced by expression of antigen in a wide range of 'amateur' APC cell types, including developing conventional T lymphocytes, thymic innate-like T cells and thymic eosinophils.

Effects of co-stimulation, antigen localization and affinity
As co-stimulation by CD80/CD86 can be important for effective negative selection for some, but not all, TCR specificities [45][46][47][48][49] , we next assessed the expression of these molecules by thymic eosinophils and innate-like T cells (Extended Data Fig. 4a).Thymic eosinophils expressed high levels of CD80 but not CD86, whereas a large fraction of thymic iNKT cells and some γδT cells displayed both co-stimulatory molecules on their surface.As data from the Immgen database 20,34 suggest that iNKT cells express little if any Cd80 or Cd86 mRNA, and as T cells often acquire CD80 and CD86 from APCs through trogocytosis 50,51 , it seems likely that iNKT cells acquire these molecules from professional thymic APCs.Addition of anti-CD80/CD86 to RTOCs containing Jedi-TCRαβ DP thymocytes and WT or IL-4-GFP iNKT cells had no effect on negative selection (Extended Data Fig. 4b), suggesting that co-stimulation might be dispensable for negative selection in the case   Frequency of GFP + cells (n = 5 mice) and pie chart showing frequencies of different subsets within the GFP + compartment (average percentage ± s.d.; n = 3 mice).See also Extended Data Fig. 2a-c.b, Images of IL-4-GFP thymus sections as analyzed by confocal immunofluorescence microscopy.The medulla was identified by staining with UEA1 lectin (blue).IL-4-GFP + eosinophils were distinguished from IL-4-GFP + lymphocytes (green) by co-staining with anti-Siglec-F (red).See also Extended Data Fig. 2d.c, WT and IL-4-GFP mice (on a BALB/c (H2 d ) background; CD45.2) were lethally irradiated and reconstituted with syngeneic WT or IL-4-GFP BM cells mixed with BM cells from Jedi mice (B10.D2 genetic background (H2 d ); CD45.1).Thymi of the chimeras were analyzed 6 weeks after reconstitution (n = 4 chimeras for analysis of thymi in WT + Jedi-TCRαβ → WT; n = 6 for the other groups).Jedi thymocytes were gated as CD45.1 + tetramer + cells.Expression of CD4 and CD8β on CD45.1 + tetramer + cells and viability dye and PD-1 staining of CD45.1 + tetramer + CD4 + CD8β + ( Jedi DP) cells are shown.The frequencies of CD4 + CD8β + (DP) and CD4 -CD8β + (CD8SP) cells of CD45.1 + tetramer + Jedi thymocytes and the frequencies of dead Jedi DP cells were quantified (bottom).Data are representative of three independent experiments; Tet, tetramer.d, Analysis of the frequency of cleaved caspase-3 + cells among tetramer + CD4 + CD8β + ( Jedi DP) cells in WT + Jedi-TCRαβ → WT and IL-4-GFP + Jedi-TCRαβ → WT mixed BM chimeras (from the experiment shown in Supplementary Fig. 2).Data are from one experiment with four chimeras per group; Casp3, caspase-3.e, Representative flow cytometric analysis of splenocytes from four groups of BM chimeras (same experiment as in c).Jedi cells were identified by H2-K d GFP 200-208 tetramer staining, and CD8α, CD8β and CD4 expression on these cells was analyzed.The frequencies of total and CD8α hi CD8β hi tetramerbinding cells among splenocytes were quantified (bottom).Data are presented as mean ± s.d.(*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) and were analyzed by two-tailed Student's t-test (d) or one-way ANOVA with a Holm-Sidak multiple comparisons test (c and e). of Jedi thymocytes.However, as the co-stimulation requirement for negative selection is not absolute and depends on the epitope 45 , and as thymic innate-like T cells are equipped with CD80 and CD86, it is conceivable that co-stimulation could contribute to the induction of tolerance to some self-antigens expressed by these cells.
As many endogenous inflammation-associated self-antigens are secreted molecules, we next tested if innate-like T cells can induce tolerance to a secreted antigen.To this end, we generated retroviral constructs encoding intracellular (i.c.GFP) and secreted (secGFP) versions of GFP and confirmed efficient GFP secretion in the latter setting (Extended Data Fig. 4c).We next transduced thymic iNKT cells with Thy1.1-IRES-i.c.GFP, Thy1.1-IRES-secGFP and Thy1.1-only retroviruses, sorted Thy1.1 + iNKT cells and added them to RTOCs together with Jedi-TCRαβ DP thymocytes.A comparable loss of Jedi cells was observed with i.c.GFP and secGFP constructs, and the remaining cells upregulated PD-1 expression and downregulated CD8 expression (Fig. 4c).
Finally, to test how antigen affinity affects negative selection, we took advantage of altered peptide ligands described for the OT-I TCR.It was shown that Q4, T4 and V4 substitutions in the SIINFEKL epitope (N4) reduced OT-I CD8 + T cell reactivity by 20-, 70-and 700-fold, respectively 52 .We therefore retrovirally transduced thymic iNKT cells with constructs encoding cytoplasmic ovalbumin (cOVA)-IRES-GFP containing these epitopes and added GFP + iNKT cells to RTOCs together with sorted OT-I DP thymocytes.N4-, Q4-and T4-expressing iNKT cells induced a near complete elimination of OT-I thymocytes (33-, 18-and 9-fold decreases, respectively), and even very low-affinity V4 epitope was sufficient to induce elimination of about half of the OT-I thymocytes (Fig. 4d).
We conclude that expression of either intracellular or secreted antigens by innate-like T cells can be sufficient to induce elimination of autoreactive CD8 + thymocytes and that this elimination can take place across a wide range of affinities.

Antigen expression by iNKT cells induces tolerance in vivo
We next sought to test if antigen expression by innate-like T cells is also sufficient to mediate tolerance induction in vivo.As a first approach to this question, we used IL-17A-GFP mice in which, in contrast to the IL-4-GFP system, GFP expression in the thymic hematopoietic compartment was restricted to innate and innate-like lymphocytes (Fig. 5a and Extended Data Fig. 5a,b).Analysis of stromal populations in these mice also revealed GFP expression in a small subset of mTECs (Extended Data Fig. 5c).Therefore, to dissect the roles of GFP-expressing stromal and hematopoietic populations, we established WT → WT, IL-17A-GFP → WT, WT → IL-17A-GFP and IL-17A-GFP → IL-17A-GFP BM chimeras and immunized these mice with the GFP 200-208 peptide.Fully mirroring the results obtained in the IL-4-GFP system (Fig. 2), IL-17A-GFP expression either by radioresistant or radiosensitive cells was sufficient to abrogate CD8 + T cell responses to GFP 200-208 (Fig. 5b), whereas no CD4 + T cell tolerance to GFP 81-95 MHC class II epitope immunization was observed in IL-17A-GFP mice (Extended Data Fig. 5d).Unexpectedly, GFP-expressing cells were very poorly reconstituted in the thymi of IL-17A-GFP → WT BM chimeras (0.009 ± 0.003% GFP + cells of total thymocytes in IL-17A-GFP → WT BM chimeras versus 0.06 ± 0.04% in steady-state IL-17A-GFP mice and 0.5 ± 0.2% in IL-4-GFP mice; Extended Data Fig. 5e).This near absence of GFP-expressing cells in the thymi of IL-17A-GFP BM chimeras precluded us from assessing negative selection of supraphysiological numbers of autoreactive GFP-specific thymocytes in Jedi:IL-17A-GFP mixed BM chimeras, a setting that in fact resulted in breach of tolerance (see below).As an alternative approach, we therefore turned to RTOC experiments, which demonstrated that iNKT cells from IL-17A-GFP mice can induce efficient elimination of Jedi-TCRαβ DP thymocytes (Fig. 5c).
The immunization experiments in IL-17A-GFP → WT BM chimeras described above showed that expression of a model inflammationassociated self-antigen solely by innate-like lymphocytes is sufficient to induce tolerance in vivo, and RTOC experiments suggested that it can happen through negative selection of autoreactive thymocytes.To directly test if exclusive antigen expression by thymic innate-like T cells is sufficient to induce inactivation of autoreactive CD8 + thymocytes in vivo, we injected sorted thymic IL-4-GFP iNKT cells into the thymi of Jedi-TCRβ mice, which, before injection, contained 0.007 ± 0.003% GFP-specific thymocytes (Fig. 5d).GFP + cells were detectable in the thymi of the recipient mice 7 days after transfer (Fig. 5d), but their frequency was much lower than that in IL-4-GFP mice (Fig. 3a) and similar to what was observed in steady-state IL-17A-GFP mice (Fig. 5a).The presence of these minute numbers of GFP + iNKT cells resulted in a decreased frequency of GFP-specific thymocytes and an acquisition of a CD4 -CD8α lo CD8β lo PD-1 hi cell-surface phenotype by the remaining GFP-specific cells (Fig. 5d).Thus, antigen expression exclusively by small numbers of thymic innate-like T cells is sufficient to induce elimination of autoreactive CD8 + thymocytes in vivo.

T cell-to-T cell antigen presentation mediates negative selection
The efficient negative selection of autoreactive thymocytes by small numbers of antigen-expressing innate-like T cells suggested possible cross-presentation by thymic professional APCs.To start exploring this possibility, we sorted DCs, iNKT cells and eosinophils from the thymi of WT, IL-4-GFP and IL-17A-GFP mice and cocultured them with an NFAT reporter cell line 53 expressing the Jedi TCR.Reporter expression was upregulated in cocultures with iNKT cells from IL-4-GFP and IL-17A-GFP mice and eosinophils from IL-4-GFP mice but not in cocultures with DCs (Extended Data Fig. 6a, note the higher baseline reporter expression in all DC cocultures), suggesting that IL-4-GFP and IL-17A-GFP may not undergo efficient cross-presentation by thymic DCs.
To directly test if cross-presentation could contribute to negative selection, we studied the fate of Jedi-TCRαβ DP thymocytes in RTOCs https://doi.org/10.1038/s41590-024-01899-6 established with iNKT cells sorted from the thymi of IL-4-GFP mice either on an H2 b/d background (capable of presentation to Jedi thymocytes) or on an H2 b/b background that makes direct presentation to Jedi thymocytes impossible.Negative selection and phenotypic changes of Jedi cells were completely abrogated when iNKT cells were unable to directly present the GFP peptide to Jedi thymocytes due to this MHC mismatch, even when such iNKT cells were present at highly supraphysiological numbers (Fig. 6a).No evidence for cross-presentation was obtained when MHC-mismatched iNKT cells from IL-17A-GFP mice or MHC-mismatched iNKT cells expressing a secreted version of GFP were used in RTOC experiments (Extended Data Fig. 6b,c).
To assess the contribution of direct versus cross-presentation of IL-4-GFP in vivo, we next established three groups of BM chimeras: Importantly, in the latter setting, more than half of the thymic DCs expressed H2-K d (Extended Data Fig. 6d) and therefore were theoretically capable of cross-presentation of GFP 200-208 epitope to Jedi cells.Although GFP-specific thymocytes were eliminated when direct presentation was possible, 'direct presentation-deficient' IL-4-GFP H2 b/b + Jedi-TCRβ H2 b/d → WT chimeras had numbers and cell-surface phenotypes of GFP-specific thymocytes indistinguishable from those of the no-antigen group (Fig. 6b).Finally, intrathymic injections of H2 b/b (no direct presentation) or H2 b/d (direct presentation is possible) iNKT cells expressing a secreted version of GFP into Jedi-TCRβ H2 b/d recipients demonstrated that PD-1 upregulation and CD8 + downregulation was evident only when transferred iNKT cells were capable of direct antigen presentation (Extended Data Fig. 6e).We conclude that in the case of GFP as an innate-like T cell-derived model antigen, it undergoes direct T cell-to-T cell presentation but not cross-presentation by thymic APCs.
Finally, to visualize these T cell-to-T cell antigen presentation events, we analyzed the interactions between fluorescently labeled iNKT cells and Jedi-TCRαβ thymocytes loaded with Calbryte-520 (Cal-520) AM Ca 2+ sensor dye in RTOCs by confocal microscopy.Jedi DP cells for these experiments were sorted for low levels of PD-1 expression to enrich TCR-expressing cells (Extended Data Fig. 6f).RTOCs were allowed to assemble overnight and were then imaged by confocal microscopy for 13 h (Fig. 6c-g).Both iNKT cells and viable Jedi-TCRαβ DP thymocytes were highly motile in these organoids (Supplementary Videos 1-4).In line with the expression of the Jedi TCR by about 70% of DP thymocytes at the beginning of the culture and their ongoing negative selection, approximately one-third of the contacts between Jedi thymocytes and IL-4-GFP iNKT cells resulted in Ca 2+ flux in Jedi-TCRαβ DP cells (Fig. 6d-f, Extended Data Fig. 6g and Supplementary Video 1).
No such contact-induced Ca 2+ signaling was observed in RTOCs with WT iNKT cells (Fig. 6d-f and Supplementary Video 2).Contacts between IL-4-GFP iNKT cells and Jedi-TCRαβ DP cells that lead to Ca 2+ signaling were longer than those that did not result in Ca 2+ flux (Fig. 6g), and, in some cases, Jedi thymocytes exhibited chasing behavior toward IL-4-GFP iNKT cells (Supplementary Video 3).In few cases, contact-induced Ca 2+ flux in Jedi-TCRαβ thymocytes was followed by a loss of motility and changes in cellular morphology suggestive of cell death (Supplementary Video 4).Thus, direct antigen presentation by iNKT cells to autoreactive thymocytes was confirmed by visualization of TCR signaling in autoreactive CD8 + thymocytes.

Lack of tolerance to IL-17A-GFP results in a fratricide reaction
We next sought to investigate the consequences of failure to disarm CD8 + T cells reactive to an antigen expressed by effector T cells.Our initial analysis of PLZF lu/lu and Tcrd -/-Cd1d -/-Mr1 -/-mice, which lack some innate-like T cell populations, did not reveal autoimmune phenotypes, in line with the unexpected abundance of all functional effector subsets in the thymi of these mice (see Supplementary Note 2 and Supplementary Figs. 3 and 4).We therefore next decided to take advantage of the fact that the thymic IL-17A-GFP + compartment was not properly reconstituted in IL-17A-GFP → WT BM chimeras, and GFP + cells were nearly absent in the thymi of these mice, whereas the peripheral IL-17A-GFP effector subsets were less affected in this setting (Extended Data Figs.5e and 7a).We hypothesized that if we tip the balance between scarce thymic IL-17A-GFP + cells and developing GFP-specific CD8 + thymocytes toward the latter cells in this system, some autoreactive CD8 + T cells should escape to the periphery and may cause autoimmune elimination of peripheral T H 17 cells.To test this hypothesis, we established the following three groups of BM chimeras: (1) IL-17A-GFP → WT (no overproduction of autoreactive T cells), ( 2) WT + Jedi-TCRβ → WT (no antigen) and ( 3) IL-17A-GFP + Jedi-TCRβ → WT (predominantly peripheral antigen and overproduction of autoreactive T cells; Fig. 7a).As expected, small numbers of GFP-specific CD8 + T cells with naive cell-surface phenotype were found in peripheral lymphoid organs of WT + Jedi-TCRβ → WT chimeras (Fig. 7b and Extended Data Fig. 7b).By contrast, lymphoid organs of IL-17A-GFP + Jedi-TCRβ → WT chimeras contained strongly expanded GFP-specific CD8 + T cells with a CD44 + PD-1 + activated cell-surface phenotype (Fig. 7b and Extended Data Fig. 7b).Thymi of IL-17A-GFP + Jedi-TCRβ → WT BM chimeras also exhibited an accumulation of GFP-specific CD44 + PD-1 + CD8 + T cells (Extended Data Fig. 7c), possibly reflecting recruitment of activated cells from the periphery.Of note, this was in stark contrast to the previous observations made in IL-4-GFP + Jedi-TCRβ → WT BM chimera thymi that exhibited a complete loss of GFP-specific cells (Fig. 6b and Supplementary Fig. 2).This accumulation of activated autoreactive CD8 + effector T cells coincided with a near complete loss of IL-17A-GFP + T H 17 cells (Fig. 7c and Extended Data Fig. 7d,e; gating on V β 4 -cells was applied to exclude Jedi cells from the analysis).These results suggest that breach in tolerance induction results in an autoimmune attack that may eliminate peripheral IL-17A-expressing effector T cells.To formally test this, we performed an in vivo killing assay by transferring T H 17 cells in vitro differentiated from CD4 + T cells of IL-17A-GFP mice.Only a fraction of these cells induced the expression of GFP (which correlated well with the production of IL-17A; Extended Data Fig. 7f) and therefore would represent possible targets for activated Jedi cells.These GFP + cells were selectively eliminated in IL-17A-GFP + Jedi-TCRβ → WT, but not in WT + Jedi-TCRβ → WT, chimeras (Fig. 7d).Together, these results demonstrate that failure to induce CD8 + T cell tolerance to a single inflammation-associated self-antigen can result in a fratricide reaction that leads to elimination of the entire class of effector T cells expressing this molecule.

Induction of tolerance to an endogenous effector molecule
Finally, we sought to test if expression of an endogenous effector T cell molecule exclusively in the hematopoietic compartment is sufficient to induce CD8 + T cell tolerance to this antigen.We focused on IFNγ, the production of which among thymocytes was restricted to T cell/ NK cell/innate lymphoid cell (ILC) lineages, as evidenced by Thy1 and NK1.1 expression and lack of MHC class II expression by IFNγ + cells (Extended Data Fig. 8a).We identified an H2-K b epitope in IFNγ (IFNγ 69-78 QIISFYLRLF), the immunization of which resulted in a strong CD8 + T cell response in Ifng -/-, but not WT, animals (Extended Data Fig. 8b).We next established the following four groups of BM chimeras: (1) WT → WT, (2) Ifng -/-→ WT, (3) WT → Ifng -/-and (4) Ifng -/-→ Ifng -/-and immunized them with IFNγ 69-78 and an irrelevant foreign peptide (ovalbumin (OVA 257-264 ) SIINFEKL).Intracellular staining for tumor necrosis factor (TNF) after a brief peptide restimulation was used as a readout  https://doi.org/10.1038/s41590-024-01899-6 for antigen-specific CD8 + T cell responses.All four groups of chimeras showed a response to the foreign epitope immunization (Extended Data Fig. 8c).By contrast, only Ifng -/-→ Ifng -/-chimeras exhibited a strong response to IFNγ 69-78 immunization, whereas all other groups of chimeras were equally tolerant to this epitope (Fig. 8).Thus, expression of IFNγ solely by hematopoietic cells can induce CD8 + T cell tolerance to this molecule.Together with the T cell/NK cell/ILC-restricted expression, these results suggest that antigen expression by these effector subsets is sufficient for induction of tolerance to this endogenous inflammation-associated self-antigen.

Discussion
Self/non-self discrimination by the adaptive immune system largely relies on the context of antigen encounter.For T lymphocytes, a variety of tolerance mechanisms ensure inactivation of autoreactive cells in the thymus or in the periphery in the absence of inflammation induced by pathogen invasion.Only if a mature T lymphocyte encounters an antigen in the context of inflammation does it receive all signals required to induce clonal expansion and execution of effector programs.The expression of numerous self-molecules involved in T cell effector programs therefore overlaps in time and space with the emergence of pathogen-derived antigens.In this study, we aimed to investigate how the immune system distinguishes such inflammation-associated self-antigens from pathogen-derived molecules.We demonstrated that CD8 + T cell tolerance to inflammation-associated self-antigens can be induced in the thymus through antigen presentation by a variety of thymic cell types.These include two cell types not previously implicated in T cell tolerance induction-thymus-resident innate-like T cells and thymic eosinophils.We showed that minute numbers of thymic innate-like T lymphocytes, cells that express self-antigens characteristic of all major T cell effector programs, were sufficient to efficiently eliminate autoreactive CD8 + thymocytes.We demonstrated that CD8 + T cell tolerance induction by innate-like T lymphocytes can be achieved without the involvement of professional APCs through direct T cell-to-T cell antigen presentation.We also provided evidence for the contribution of effector lymphocytes to the induction of tolerance to an endogenous inflammation-associated self-antigen.Finally, we showed that decreased abundance of a model inflammation-associated self-antigen in the thymus can result in a spontaneous fratricide reaction, causing complete elimination of peripheral effector T cell subsets expressing this antigen.This unprovoked and extremely efficient elimination of a whole class of effector T cells after breach of tolerance to a single inflammationassociated self-antigen highlights the ultimate importance of tolerance to this group of molecules.The importance of this tolerance seems to be further reflected by the high redundancy of cell types that express such antigens in the thymus.Such redundant expression was also characteristic for IFNγ that was expressed by thymic innate and innate-like lymphocytes and a small group of TECs.Well in line with our observations with model antigens, both radioresistant and radiosensitive cells were able to mediate CD8 + T cell tolerance to this endogenous inflammation-associated self-antigen.Together with the T cell/NK cell/ILC-restricted expression of IFNγ in the hematopoietic compartment, these results provide evidence for the role of effector lymphocytes in the induction of tolerance to this proinflammatory cytokine.Although by themselves these experiments cannot discriminate between central and peripheral tolerance mechanisms, the abundance of IFNγ-expressing cells in the thymus and our results obtained with model antigens suggest that central tolerance is likely to contribute to the neutralization of IFNγ-reactive CD8 + T cells.
Although cytotoxic responses to inflammation-associated molecules have not, to our knowledge, been studied in the context of autoimmunity, many human autoimmune diseases are associated with autoantibodies against a broad range of proinflammatory cytokines 54 , suggesting that CD4 + T cell tolerance to these self-antigens is broken in such patients.Interestingly, in our study using IL-4-GFP and IL-17A-GFP as model inflammation-associated self-antigens, we observed profound CD8 + , but not CD4 + , T cell tolerance.We also did not find

Article
https://doi.org/10.1038/s41590-024-01899-6 any evidence of GFP cross-presentation by thymic DCs in this system, even when innate-like T cells expressed a secreted form of GFP.However, we did not test if secreted GFP may be subjected to MHC class II presentation by professional APCs.Moreover, the situation may be different for some endogenous secreted antigens, for example, if professional thymic APCs would express a receptor for such a molecule.It remains to be investigated if in these scenarios secreted inflammation-associated self-antigens would be taken up and presented by DCs, enabling the induction of CD4 + T cell tolerance.Alternatively, it is also possible that induction of CD4 + T cell tolerance is less stringent for inflammation-associated self-antigens, making them frequent targets in autoimmunity.Finally, unlike their mouse counterparts, human thymocytes can express MHC class II molecules 55 , and the expression of genes encoding MHC class II chains was relatively high in some of the thymocyte populations that expressed T cell effector genes (Extended Data Fig. 1).It is therefore conceivable that in humans, these cells could also present antigens to developing CD4 + thymocytes and therefore contribute to the induction of CD4 + T cell tolerance.
Although the role of direct antigen presentation by professional APCs in the elimination of autoreactive thymocytes is well documented 8,10 , the contribution of antigen presentation by nonprofessional APCs to this process under physiological circumstances remained unclear.In in vitro systems, peptide-pulsed thymocytes are capable of direct antigen presentation and induction of cell death in autoreactive thymocytes 56,57 .In an in vivo setting, mature T cells were shown to mediate the induction of central tolerance to an alloantigen 58 .In addition, it was demonstrated that autoantigens expressed by T cells 59 and thymic fibroblasts 60 can contribute to the induction of T cell tolerance.However, it remains unclear if the latter happens through direct antigen presentation of autoantigens or through their cross-presentation by professional APCs.
In this study, we have demonstrated that endogenous antigen expression by minute numbers of thymic-resident innate-like T cells, thymic eosinophils and conventional developing thymocytes was sufficient to eliminate autoreactive CD8 + thymocytes.Moreover, we demonstrated that innate-like T cells can induce CD8 + T cell tolerance by directly presenting self-antigens to autoreactive thymocytes.These results substantially expand the array of known cell types that can mediate the induction of T cell tolerance and suggest that most cell types that express MHC class I and are present in the thymus are likely able to contribute to the induction of CD8 + T cell tolerance.In addition to B cells and eosinophils, mouse and/or human thymi were reported to harbor several NK cell and ILC subsets, plasma cells, mast cells, erythroblasts, megakaryocytes and recirculating memory T cells 35,[61][62][63] .Thus, in addition to ectopic antigen expression by mTECs, recruitment and/or retention of these 'ectopic cell types' may represent a mechanism that ensures representation of hematopoietic system-derived self-antigens in the thymus.
Recent studies have demonstrated that large fractions of iNKT cells and γδT cells in the thymus are tissue-resident cells 23,24 .Moreover, thymus-resident innate-like T cells are in the center of a complex 'ecosystem' that actively ensures their presence in the thymus and production of inflammatory mediators by these cells.Indeed, a tuft cell-like subset of mTECs is required for iNKT cell accumulation in the thymus 5 , whereas production of IL-4 by these cells is ensured by their CD1d-dependent activation by thymic myeloid cells 64 .In turn, type 2 cytokines secreted by iNKT cells support the intrathymic generation of IFNγ-producing CD8 + T cells 25,26 , regulate the recruitment of thymic eosinophils 27 and induce the activation thymic DCs 29 .In addition, proinflammatory cytokines of both stromal (type 1 IFNs, IL-15, IL-6 and thymic stromal lymphopoetin) and innate-like T cell (IFNγ) origin were shown to regulate thymic T cell development 65,66 , indicating that these mediators are present in the thymic microenvironment in biologically meaningful amounts.Moreover, several inflammatory pathways, including type 1/type 3 IFN signaling and Toll-like receptor and/or IL-1β signaling, are constitutively active in TECs 30,31 .
In this study, we demonstrated that at least two cellular components of this inflammatory network (thymic innate-like T cells and eosinophils) were sufficient to mediate the induction of remarkably efficient tolerance to antigens that they express.Taking these observations together, it is tempting to speculate that constitutive activation of a plethora of inflammatory pathways in the thymus may have evolved to ensure induction of tolerance to inflammation-associated self-antigens, a class of molecules that otherwise largely mirror the spatial and temporal distribution of pathogen-derived antigens.

Flow cytometry
Mouse spleen tissue, lymph nodes and thymi were collected, and single-cell suspensions were obtained by mincing through 70-µm cell strainers.Isolation of intestinal intraepithelial lymphocytes (iIELs) was performed as described previously 72 .Isolation of TECs was performed as described previously 73 with minor modifications.In short, each thymus was digested in 0.5-1 ml of RPMI 1640 medium with 1 mg ml -1 collagenase IV and 100 U ml -1 DNase I for 20 min at 37 °C before staining with APC-labeled anti-EPCAM, followed by enrichment using anti-APC microbeads (Miltenyi Biotec) and magnets from the EasySep Cell Isolation and Separation Technology platform (STEMCELL Technologies).For detection of antigen-specific T cells, single-cell suspensions were incubated with APC-and/or PE-conjugated H2-K d GFP 200-208 or I-A b GFP 81-95 , PE-or BV421-conjugated CD1d-PBS-57 or APC-conjugated MR1-5-OP-RU tetramers (all provided by the National Institutes of Health tetramer core facility) for 1 h before enrichment and further cell-surface staining (for I-A b GFP 81-95 ) or they were directly included into the surface antibody staining mix (for all other tetramers).Tetramer-binding CD4 + T cells were enriched by magnetic enrichment as described by Moon et al. 74 .Dead cells were detected using a Live/Dead Fixable Aqua Dead Cell Stain kit for 405-nm excitation (Thermo Fisher Scientific) or the fixable viability dye eFluor780 (eBioscience) according to manufacturers' instructions and were excluded from further analysis unless stated otherwise.
For the detection of cleaved caspase-3, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Bioscience) and stained with rabbit anti-mouse active caspase-3 (5A1E) on ice for 30 min, followed by incubation with PE-conjugated anti-rabbit IgG (H + L) F(ab′) 2 fragment antibody on ice for 30 min.Intracellular cytokine staining was performed using a BD Cytofix/Cytoperm kit (BD Bioscience) following manufacturer's instructions.
Data were acquired on an LSR Fortessa, FACSCanto II (BD Biosciences) or Cytek Aurora (Cytek Biosciences) flow cytometer and analyzed with FlowJo software v.10 (BD Biosciences).Cell sorting was performed using FACS Aria III and FACS Fusion cell sorters (BD Biosciences).

Generation of BM chimeras
For all BM chimera experiments, BM cells from donor mice were stained with APC-labeled CD4, CD8, CD3, TCRβ, TCRγδ, NK1.1 and Ter119 antibodies, followed by magnetic depletion of T and NK cells with anti-APC microbeads (Miltenyi Biotec).BM cells (2 × 10 6 -5 × 10 6 ) were then transferred i.v.into lethally irradiated (split dose of 5 Gy, twice, using an Xstrahl CIX3 X-ray irradiator) recipients.For mixed BM chimera experiments shown in Fig. 3c, Jedi-TCRαβ BM cells were mixed with WT or IL-4-GFP BM cells at a 3:1 ratio before transfer.For mixed BM chimera experiments shown in Fig. 6b, Jedi-TCRβ H2 b/d BM cells were mixed at a 1:1 ratio with IL-4-GFP H2 b/b or IL-4-GFP H2 b/d BM cells before transfer.For mixed BM chimera experiments shown in Fig. 7, Jedi-TCRβ BM cells were mixed at a 1:20 ratio with IL-17A-GFP or WT BM cells before transfer into WT recipients (all on a CB6F1 genetic background).For mixed BM chimera experiments shown in Supplementary Fig. 2, Jedi-TCRβ or Jedi-TCRαβ BM cells were mixed at a 1:1 ratio with IL-4-GFP or WT BM cells before transfer into WT recipients (all on a BALB/c genetic background).

In vivo killing assays
In vitro-differentiated IL-17A-GFP T H 17 cells were labeled with 1 µM CTV (Thermo Fisher) in PBS with 0.5% bovine serum albumin (BSA) in a water bath at 37 °C for 8 min.Labeled cells were washed and i.v.transferred (0.5 × 10 6 ) into the indicated groups of BM chimeras.Disappearance of GFP hi CTV + cells was assessed by flow cytometry in the spleens of the recipient mice 20 h after transfer.
In vitro-differentiated T H 2 cells were labeled with 1 µM CTV and mixed with 10 µM CTV-labeled freshly isolated naive T cells at a 1:3 https://doi.org/10.1038/s41590-024-01899-6ratio.Labeled cells were washed with PBS and i.v.transferred into the indicated groups of BM chimeras (2 × 10 6 cells per mouse).The ratio between CTV int (T H 2) and CTV hi (naive) cells was assessed in the spleens of recipient mice 20 h after transfer by flow cytometry.

Transfection and retrovirus production
HEK293T cells were cultured in DMEM supplemented with 10% FCS, 50 µM 2-mercaptoethanol, 2 mM l-glutamine, 100 U ml -1 penicillin and 100 U ml -1 streptomycin at 37 °C and 5% CO 2 until the confluency reached 70%.To test GFP expression and secretion, HEK293T cells were transiently transfected with Thy1.1-IRES-i.c.GFP or Thy1.1-IRES-secGFP constructs by calcium phosphate transfection, as previously described 77 .The culture medium was replaced with DMEM (without phenol red) 6 h after transfection.Supernatants were collected 8, 24 and 48 h after transfection, and fluorescence intensity was measured with a Varioskan LUX Multimode Microplate Reader (Thermo Fisher) at an excitation wavelength of 480 nm and emission wavelength of 520 nm (with a bandwidth of 12 nm).
Retrovirus-containing supernatants were generated by transient cotransfection of HEK293T cells with the retroviral constructs described above and pCL-Eco packaging vector using calcium phosphate transfection.Viral supernatants were collected 48 h after transfection, mixed thoroughly with 5× PEG solution (40% polyethylene glycol 10,000 (Sigma) and 2.4% NaCl dissolved in water) and incubated overnight at 4 °C.The next day, virus particles were precipitated by centrifugation at 2,000g and 4 °C for 1 h, resuspended in fresh culture medium to reach 100-times the concentration and stored at −80 °C.
For CD4 + T cell peptide immunizations, mice were subcutaneously injected with 100 µl of an emulsion containing 100 µg of GFP 81-95 peptide (HDFFKSAMPEGYVQE, I-A b epitope; Genscript).Emulsions were prepared by mixing the peptide in PBS in a 1:1 ratio with complete Freund's adjuvant (Sigma-Aldrich).
For detection of antigen-specific CD8 + responses in peptideimmunized Ifng -/-/WT BM chimeras, spleens were collected 6 days after immunization and processed to a single-cell suspension.After erythrocyte lysis, splenocytes were resuspended in IMDM containing the indicated peptides (final concentration of 10 µg ml -1 ) and cultured in a six-well plate at a density of 2 × 10 6 cells per ml (1 × 10 7 cells per well) at 37 °C and 5% CO 2 .After a 1-h incubation, brefeldin A (3 µg ml -1 ) was added to the culture.Cells were collected, stained as described above and analyzed 5 h after the addition of brefeldin A. Antigen-specific CD8 + T cells were identified as TCRβ + CD8 + CD44 hi TNF + in Fig. 8 and Extended Data Fig. 8b,c.

Generation of the Jedi TCR-expressing reporter cell line
NFAT-sFT reporter-containing 16.2c11 cells 53 (a kind gift of J. Kisielow, Repertoire Immune Medicines) were retrovirally transduced with constructs encoding Jedi TCR, CD8α and CD8β.Retroviral transfection was performed as described previously 77 .Cells were sorted and expanded to generate a CD8α + CD8β + Jedi TCR-expressing reporter cell line.

Retroviral infection of thymic iNKT cells
Single-cell suspensions of thymocytes were stained with APC-labeled antibodies to CD24 and CD8 (anti-CD11c was added for the experiment shown in Extended Data Fig. 6e), followed by magnetic depletion with anti-APC microbeads (Miltenyi Biotec) to enrich for iNKT cells.Enriched thymic iNKT cells were cultured at a density of 1 × 10 6 cells per ml in IMDM supplemented with 10 ng ml -1 IL-7 (Peprotech), 100 ng ml -1 IL-15 (Peprotech), and 0.5 µg ml -1 CD1d-PBS-57 tetramer.After 2 days, iNKT cells were centrifuged at 400g at room temperature for 5 min and resuspended in fresh IMDM containing 10 ng ml -1 IL-7, 100 ng ml -1 IL-15, 4 µg ml -1 polybrene (Sigma) and 20 µl ml -1 concentrated virus particles, transferred to plates precoated with 20 µg ml -1 RetroNectin (Takara) and spin infected at 1,000g and 32 °C for 1 h.One more round of https://doi.org/10.1038/s41590-024-01899-6infection was performed 6 h after the first round of infection.Eighteen hours after the second round of infection, the culture medium was replaced with fresh IMDM containing 10 ng ml -1 IL-7 and 100 ng ml -1 IL-15.Forty-eight hours after the first round of infection, iNKT cells were used in RTOC and intrathymic transfer experiments.

Immunofluorescence microscopy of thymus sections
Organ sections were prepared as described previously 78 .For GFP detection, slides were incubated with Alexa Fluor 488-conjugated GFP booster (Chromotek), and staining was performed according to the manufacturer's instructions.For mTEC detection, slides were incubated with biotinylated UEA1 (Vector Laboratories) followed by incubation with APC-conjugated streptavidin (BioLegend) according to the manufacturer's instructions.Confocal images were acquired on an LSM 800 system (Carl Zeiss) at the Biomedicum Imaging Core at the Karolinska Institute.Images were processed using Zen 2.3 Black Edition (Carl Zeiss) or Imaris (Bitplane) imaging software.

RTOC live-cell imaging
For live-cell imaging, RTOCs were prepared as described above with minor modifications.Jedi DP thymocytes (gated as CD4 + CD8 + PD-1 lo/int cells) isolated from the thymi of Jedi mice and iNKT cells (gated as GFP + CD1d tetramer-binding cells) from the thymi of IL-4-GFP mice were sorted.After sorting, Jedi DP thymocytes were loaded with 5 µM Cal-520 AM (AAT Bioquest) in HBSS containing 2% FCS for 1 h at 37 °C.DP Jedi cells were then washed twice with FCS-containing RPMI 1640 culture medium and labeled with 2.5 µM CTV (Thermo Fisher) in PBS/0.5% BSA for 8 min in a water bath at 37 °C, while thymic iNKT cells were labeled with 2.5 µM CTY (Thermo Fisher) under the same labeling conditions.RTOCs were allowed to assemble for 16-20 h before transfer to compartmentalized glass-bottom dishes (ibidi).For imaging, RTOCs were first embedded in PureCol EZ Gel solution (Sigma-Aldrich) for 45 min at 37 °C.RTOC complete medium was then added on top of the collagen matrix.z-stacks of two RTOCs were first imaged for each condition at a rate of 1 frame per min for 1-3 h using a ×20/0.8-NAPlan-Apochromat objective and a fast Airyscan detector on an LSM880 microscope (Carl Zeiss) with the incubation chamber set to 37 °C and 5% CO 2 .Afterward, longer acquisitions of 12 h at 1 frame per min were conducted using standard confocal settings.

RTOC live-cell imaging analysis
Maximum intensity projections of confocal datasets were generated and analyzed in Zen Lite software (Carl Zeiss).Cell contacts were defined by colocalization of an iNKT cell and a Jedi thymocyte for at least three frames (one frame = 1 min).Contact identified using maximum intensity projections was then validated using the full three-dimensional datasets.Consecutive detachment and attachment between two cells with short intervals (shorter than five frames) were counted as one contact.In total, 134 contacts (for RTOCs with WT iNKT cells) and 108 contacts (for RTOCs with IL-4-GFP iNKT cells) were analyzed manually for an increase in Cal-520 AM fluorescence to enumerate Ca 2+ signaling events (Fig. 6e).For quantification of contact length, the number of frames where cell colocalization had been observed was counted.Contacts where cells could not be tracked throughout the entire contact (for example, cells masked by other cells and cells disappearing from the field of view) were excluded from this analysis.Contacts lasting for 2 frames or less were also excluded from quantification of contact length.
For quantification of Cal-520 AM signal intensity over time, ten contacts from each group (WT iNKT cells, IL-4-GFP iNKT cells with Ca 2+ signaling detected and IL-4-GFP iNKT cells with no Ca 2+ signaling detected) were randomly selected and analyzed in ImageJ.The Cal-520 AM signal intensity and CTV signal intensity in Jedi cells were quantified by measuring the mean intensity of each signal in the region of interest (ROI; corresponding to a Jedi cell) drawn manually for each frame.For each frame, Ca 2+ signal intensity of the Jedi cell was calculated as Ca 2+ signal intensity (per frame) = (mean intensity of Cal-520 AM in ROI)/ (mean intensity of CTV in ROI).The baseline Ca 2+ signal intensity for each Jedi cell was then calculated by averaging the Ca 2+ signal intensity (per frame) for five frames right before contact initiation.Normalized Ca 2+ signal intensities plotted in Fig. 6f and Extended Data Fig. 6g were calculated for each frame using the following equation: normalized Ca 2+ signal intensity = (Ca 2+ signal intensity (per frame))/(baseline Ca 2+ signal intensity).

Autoantibody profiling using bead-based protein microarrays
Serum from PLZF lu/lu mice was tested for the presence of antibodies against autoantigens and proteins from pathogens.A custom microbead-based antigen array was created to profile serum samples for antibodies against cytokines, autoimmune-associated antigens and viral antigens, as previously described 80 .The array was constructed by conjugating antigens to uniquely barcoded carboxylated magnetic beads (MagPlex-C, Luminex).Beads were qualified using prototype plasma or serum samples with known reactivities.Although the available array was constructed using human antigens, protein homology with mouse antigens still allowed for comparisons in mouse autoantibody development 81,82 .Mouse serum samples were tested at a dilution of 1:100 in 0.05% PBS-Tween supplemented with 1% (wt/vol) BSA.Bound antibody was detected using R-PE-conjugated Fcγ-specific goat anti-mouse IgG F(ab′) 2 fragment ( Jackson ImmunoResearch, 115-116-071) before analysis using a FlexMap3D instrument (Luminex).Binding events were displayed as median fluorescence intensity (MFI).For normalization, the MFI value for the 'bare bead' ID (no conjugated antigen) was subtracted from the MFI value for the antigen-conjugated bead ID.Samples were run in duplicate.Serum samples from MR/Fas lpr/lpr mice (a mouse model for systemic lupus erythematosus) served as positive controls.

Analysis of public expression datasets
AnnData objects containing previously described human and mouse thymus cell atlases 35 were downloaded from https://developmental.cellatlas.io/thymus-developmentand were replotted using Scanpy 83 (version 1.9.1).The combined human effector T cell signature used in Extended Data Fig. 1 was generated using the sc.tl.score_genesScanpy function and the following list of effector program genes: IL13, IL-17A, IL17F, IFNG, GZMB and GZMK.Analysis of the Immgen dataset was described previously 33 .

Statistical analysis
Statistical analyses were performed with GraphPad Prism 9 software.Error bars represent standard deviation of biological replicates (or individual RTOCs) unless stated otherwise.Two-tailed unpaired Student's t-tests or two-tailed Mann-Whitney tests were used to assess the statistical significance of one observed parameter between two experimental groups.An unpaired ANOVA with a Holm-Sidak multiple comparisons test or a Kruskal-Wallis test was used when more than two experimental groups were compared.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | Thymic innate-like T cells express a broad spectrum of inflammationassociated self-antigens.a, Expression of Il4, Ifng and Gzmb by mouse thymic cell subsets.Data are from the Immgen database (microarray dataset).

Fig. 2 |
Fig. 2 | Redundant role of radioresistant and radiosensitive cells in the induction of CD8 + T cell tolerance to a model inflammation-associated self-antigen.a, WT and IL-4-GFP mice on a C57BL/6J × BALB/c F1 (CB6F1) genetic background were immunized intraperitoneally with GFP 200-208 peptide using anti-CD40 and poly(I:C) as adjuvant or were left unimmunized (WT only).Six days after immunization, splenic H2-K d GFP 200-208 tetramer-binding CD8 + T cells were quantified by flow cytometry.Representative flow cytometry plots gated on CD8 + CD44 + T cells (left) and quantification of frequencies (from total CD8 + T cells) and absolute numbers (right) are shown (n = 5 mice per group).The data are representative of four independent experiments.b, WT and IL-4-GFP mice on a CB6F1 genetic background were immunized subcutaneously with GFP 81-95 peptide in complete Freund's adjuvant or were left unimmunized (WT only).Fourteen days after immunization, I-A b GFP 81-95 tetramer-binding cells were magnetically enriched from pooled spleens and lymph nodes and quantified by flow cytometry.Representative flow cytometry plots gated on CD4 + T cells (left) and absolute numbers of CD4 + CD44 + tetramer + T cells (right) are shown (n = 2 mice for nonimmunized, n = 5 for the other groups).Data are representative of two independent experiments; LN, lymph node.c, WT and IL-4-GFP mice (on a CB6F1 background) were lethally irradiated and reconstituted with syngeneic WT or IL-4-GFP BM cells depleted of T and NK cells.The resulting four groups of BM chimeras were immunized with GFP 200-208 peptide ≥7 weeks after reconstitution and analyzed 6 days after immunization, as described in a. WT CB6F1 mice were used as an unimmunized control.Representative plots demonstrating tetramer binding by CD8 + CD44 + T cells (left) and quantification of the frequency of CD8 + CD44 + tetramer + cells from total CD8 + T cells and their absolute numbers (right) are shown (n = 6 mice for nonimmunized, n = 7 for WT → WT, n = 7 for IL-4-GFP → WT, n = 5 for WT → IL-4-GFP and n = 6 for IL-4-GFP → IL-4-GFP).Results are pooled from two independent experiments.Data are presented as mean ± s.d.(NS, not significant (P > 0.05); *P < 0.05 and ****P < 0.0001) and were analyzed by two-tailed Student's t-test (b) or one-way analysis of variance (ANOVA) with a Holm-Sidak multiple comparisons test (a and c).

c,
Thymic iNKT cells were transduced with Thy1.1-IRES-i.c.GFP, Thy1.1-IRES-secGFP or Thy1.1-only retroviruses.Sorted Thy1.1 + iNKT cells were added to RTOCs together with Jedi-TCRαβ DP thymocytes.Flow cytometric analysis and quantification is shown as in b (n = 5 RTOCs for empty vector, n = 10 for i.c.GFP and n = 9 for secGFP).Results are pooled from two independent experiments.d, Thymic iNKT cells were transduced with cOVA-IRES-GFP retroviruses encoding the indicated versions of the OVA 257-264 epitope.Sorted GFP + iNKT cells were added to RTOCs together with OT-I DP thymocytes.RTOCs with nontransduced iNKT cells were used as a no-antigen control.Flow cytometric analysis and quantification is shown as in b (n = 6 RTOCs for uninfected, n = 3 for N4, n = 6 for Q4, n = 3 for T4 and n = 5 for V4).Representative results from two independent experiments are shown.Data are presented as mean ± s.d.(*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) and were analyzed by two-tailed Student's t-test (b) or one-way ANOVA with a Holm-Sidak multiple comparisons test (c and d).

Fig. 5 |
Fig. 5 | Expression of antigen by innate-like T cells is sufficient to mediate induction of tolerance in vivo.a, GFP expression by thymic nonstromalpopulations in IL-17A-GFP mice (as in Fig.3a; n = 5 mice).See also Extended Data Fig.5a-c; MAIT, mucosal-associated invariant T. b, Reciprocal BM chimeras were established and immunized with GFP 200-208 peptide as in Fig.2cbut using IL-17A-GFP donor and recipients (all mice were on a CB6F1 background).WT mice were used as an unimmunized control.Tetramer binding by CD8 + CD44 + T cells, frequencies of CD8 + CD44 + tetramer + cells from total CD8 + T cells and their absolute numbers are shown.Results were pooled from two independent experiments (WT → WT and IL-17A-GFP → WT groups) or from one experiment (the rest; n = 4 mice for nonimmunized, n = 8 for WT → WT, n = 11 for IL-17A-GFP → WT, n = 3 for WT → IL-17A-GFP and n = 3 for IL-17A-GFP → IL-17A-GFP).c, RTOCs with sorted Jedi DP thymocytes and thymic iNKT cells were established and analyzed as in Fig.4bbut using IL-17A-GFP iNKT cells (sorted as CD1d tetramer-binding cells (without gating on GFP); n = 2 RTOCs for WT iNKT cells and n = 3 for IL-17A-GFP iNKT cells).Representative results of three independent

Fig. 6 |
Fig. 6 | Innate-like T cells can mediate elimination of autoreactive CD8 + thymocytes through direct T cell-to-T cell antigen presentation.a, RTOCs with sorted Jedi-TCRαβ DP thymocytes and iNKT cells were established and analyzed as in Fig. 4b but using iNKT cells sorted from WT mice (H2 b/d background) or IL-4-GFP mice on H2 b/d and H2 b/b backgrounds.Results from one experiment (n = 2 RTOCs per group) are shown.b, CB6F1 WT recipient mice (H2 b/d ) were irradiated and transferred intravenously (i.v.) with a mix of BM cells from Jedi-TCRβ mice on a CB6F1 background (H2 b/d ) and WT H2 b/d , IL-4-GFP H2 b/d or IL-4-GFP H2 b/b mice (on mixed C57BL/6J and BALB/c genetic backgrounds).Thymi of chimeras were analyzed 6 weeks after reconstitution.Representative plots demonstrate the frequencies of GFP-expressing thymocytes, frequencies of H2-K d GFP 200-208 tetramer-binding thymocytes (quantification shown on the right side) and expression of the indicated cell-surface markers by the latter cells (n = 3 chimeras per group).Data are representative of two independent experiments.c, Schematic representation of RTOC experiments shown in d-g; O/N, overnight.d-g, Confocal microscopy analysis of RTOCs assembled with sorted Jedi-TCRαβ DP thymocytes (labeled with CellTrace Violet (CTV) Elimination of endogenous GFP + cells (d) In vivo killing of transferred GFP + T H

Fig. 7 |
Fig. 7 | Decreased thymic abundance of IL-17A-GFP results in autoimmune elimination of peripheral GFP + T H 17 cells.a, Schematic representation of experiments shown in b-d.WT recipient mice were irradiated and transferred i.v. with T cell-and NK cell-depleted BM cells from IL-17A-GFP mice or Jedi-TCRβ mice and WT mice or Jedi-TCRβ mice and IL-17A-GFP mice (all on a CB6F1 background).Spleens were analyzed 8 weeks after reconstitution.Note the near absence of thymic IL-17A-GFP-expressing cells in BM chimeras (see Extended Data Fig. 7a).b, Frequency and cell-surface phenotype of H2-K d GFP 200-208 tetramer-binding CD8 + T cells in the spleens of BM chimeras.c, Frequency of

Fig. 8 |
Fig. 8 | Expression of IFNγ solely by hematopoietic cells is sufficient to induce CD8 + T cell tolerance.WT and Ifng -/-mice on a C57BL/6J background were lethally irradiated and reconstituted with syngeneic WT or Ifng -/-BM cells depleted of T and NK cells.Eleven weeks after reconstitution, the resulting four groups of BM chimeras were immunized with IFNγ 69-78 and OVA 257-264 peptides using anti-CD40 and poly(I:C) as adjuvant (see also Extended Data Fig.8c).For quantification of antigen-specific CD8 + T cell responses, splenocytes were

6 CD1dExtended Data Fig. 2 |Extended Data Fig. 2 |Extended Data Fig. 3 |
program-related genes MHC class II-encoding genes Extended Data Fig. 1 | Human thymic innate and innate-like lymphocytes express a broad spectrum of inflammation-associated self-antigens.UMAP highlighting the expression of combined effector T cell gene signature (generated as describe in Methods) (top) and UMAPs highlighting the expression of the indicated genes in the human thymus cell atlas scRNA-seq dataset.https://doi.org/10.1038/s41590-024-01899-Seenext page for caption.Characterization of GFP-expressing cell types in IL-4-GFP thymi.a. Gating strategy for characterization of GFP-expressing cell types in IL4-GFP thymi.b.Frequency of GFP + cells in the indicated thymic subsets in IL4-GFP mice (n = 3 mice).c.Frequency of GFP + cells among mTECs (gated as EPCAM + CD45 -UEA1 + Ly51 -) in IL4-GFP mice (n = 3 mice).d.Images of IL4-GFP and WT thymi sections as analyzed by confocal immunofluorescence microscopy as in Fig. 3b.An area of the same image as in Fig. 3b is shown for the IL4-GFP mouse.e. Frequency of GFP + hematopoietic cells in the thymi of mixed BM chimeras shown in Fig. 3c, e.Quantification (left) and representative flow cytometric plots (right) are shown (n = 4 chimeras for [WT+Jedi-TCRαβ]→WT thymi, n = 6 for the other groups).Data are presented as the mean ± s.d.Antigen expression exclusively by small numbers of conventional thymocytes or thymic eosinophils is sufficient to induce elimination of autoreactive thymocytes.a. Schematic representation of RTOC experiments shown in b-c.b-c RTOCs with Jedi-TCRαβ DP thymocytes established and analyzed as in Fig. 4a, b but with WT or Actb-GFP CD4SP thymocytes (b) and WT or IL4-GFP eosinophils (c) used as antigen source.Representative plots showing frequencies of GFP-expressing cells, frequencies of H2-K d GFP 200-208 tetramer-binding Jedi thymocytes (quantification shown on the right) and expression of the indicated cell surface markers by the latter cells.Representative results of three independent experiments (b) (n = 2 RTOCs for WT CD4SP, n = 5 for Actb-GFP CD4SP) or results of one experiment (c) (n = 2 for WT eosinophils, n = 3 for IL4-GFP eosinophils).Data are presented as the mean ± s.d.https://doi.org/10.1038/s41590-024-01899-6

Extended Data Fig. 4 |Extended Data Fig. 4 |
See next page for caption.Assessment of possible effects of co-stimulation on induction of tolerance by innate-like T cells.a. https://doi.org/10.1038/s41590-024-01899

Extended Data Fig. 5 |Extended Data Fig. 5 |
See next page for caption.Characterization of GFP-expressing cell types in IL17A-GFP thymi.a. Gating strategy for characterization of GFP-expressing cell types in IL17A-GFP thymi.b.Frequency of GFP + cells in the indicated thymic subsets in IL17A-GFP mice (n = 5 mice).c.Frequency of GFP + cells among mTECs (gated as EPCAM + CD45

Extended Data Fig. 6 |Extended Data Fig. 7 |Extended Data Fig. 7 |Extended Data Fig. 8 |
b/b iNKT [no direct presentation] H-2K d/d iNKT [direct presentation] with IL4-GFP iNKT; Ca 2+ signaling detected RTOC with IL4-GFP iNKT; no Ca 2+ signaling detected RTOC with WT iNKT g Extended Data Fig. 6 | See next page for caption.Direct presentation by model antigen-expressing populations rather than cross-presentation by professional APCs is responsible for elimination of autoreactive GFP-specific CD8 thymocytes.a. iNKT cells, eosinophils and DCs were sorted from thymi of WT, IL4-GFP and IL17A-GFP mice and co-cultured overnight with slow fluorescent timer (sFT) NFAT reporter-containing 16.2c11 cells that were engineered to express the Jedi TCR, CD8α and CD8β.Activation of NFAT signaling was measured by sFT-Blue reporter upregulation.b.RTOCs with sorted Jedi DP thymocytes and iNKT cells were established and analyzed as in Fig. 6a, but using iNKT cells sorted from WT mice (on H2 b/d background) or from IL17A-GFP mice on H2 b/d and H2 b/b backgrounds.Results of one experiment (n = 3 RTOCs for IL17A-GFP H-2 b/b group, n = 4 for the other groups).c.Thymic iNKT cells from H2 d/d (BALB/c background) or H2 b/b (C57BL/6J background) mice were transduced with Thy1.1-IRES-i.c.GFP, Thy1.1-IRES-secGFP and Thy1.1-only retroviruses.Sorted Thy1.1 + iNKT cells were added to RTOCs together with Jedi-TCRαβ DP thymocytes.Flow cytometric analysis and quantification are shown.Results of one experiment (n = 6 RTOCs for H-2 d/d i.c.GFP and H-2 d/d secGFP groups, n = 7 for the other groups).d.Mixed BM chimeras were established as described in Fig. 7b.Expression of H-2K b and H2-K d on thymic DCs (gated as CD11c + MHC II + ) was analyzed by flow cytometry.e. Thymic iNKT cells from H2 b/d (CB6F1 background) or H2 b/b (C57BL/6J background) mice were transduced with Bcl2-P2A-Thy1.1-IRES-secGFPencoding retroviruses and injected into the thymi of Jedi-TCRβ mice (on CB6F1 background).Expression of CD8α, CD8β and PD1 on CD4 -H2-K d GFP 200-208 tetramer-binding thymocytes is shown 7 days after transfer.f.Gating strategy showing utilization of low levels of PD1 as a surrogate marker for TCR expression on DP thymocytes from Jedi-TCRαβ mice.g.Normalized Ca 2+ signaling intensity in Jedi thymocytes interacting with iNKT cells as in Fig. 6f, but values for individual cells are shown.Data are presented as the mean ± s.d. with NS: nonsignificant (P > 0.05) and **P < 0.01.Data were analyzed by one-way ANOVA with Holm-Sidak's multiple comparisons test.See next page for caption.Decreased thymic abundance of IL17A-GFP results in autoimmune elimination of GFP + T H 17 cells.a.Comparison of the frequencies of GFP + lymphocytes in the thymi, spleens and mLNs of IL17A-GFP mice and in IL17A-GFP→WT BM chimeras (analyzed 8 weeks after reconstitution).One experiment with 2 IL17A-GFP mice and 3 BM chimeras.b-e.WT recipient mice were irradiated and transferred i.v. with T-and NK-cell depleted BM cells from either IL17A-GFP mice, or Jedi-TCRβ mice and WT mice, or Jedi-TCRβ mice and IL17A-GFP mice (all on CB6F1 background).Mesenteric lymph nodes (mLN) (b, d) and thymi (c, e) were analyzed 8 weeks after reconstitution.Same experiment as in Fig. 7a-c.b, c.Frequency and cell surface phenotype of H2-K d GFP 200-208 tetramer-binding CD8 T cells.d, e. Frequency of IL17A-GFP expressing cells among total lymphocytes and among Vβ4 -(non-Jedi) CD4 T cells.Representative results of two independent experiments (n = 3 chimeras per group).f.T H 17 cells were differentiated in vitro from WT and IL17A-GFP CD4 T cells as in Fig. 7d.Intracellular expression of IL17A cytokine and GFP was analyzed after a shortterm PMA/Ionomycin restimulation.Data are presented as the mean ± s.d. with *P < 0.05 and **P < 0.01.Data were analyzed by two-tailed Student's t test.IFNγ as an endogenous T/NK/ILC-derived inflammation-associated self-antigen.a. Expression of Thy1, NK1.1 and MHC II by IFNγ + and total WT thymocytes after PMA/Ionomycin stimulation.b.WT and Ifng -/-mice (on C57BL/6J background) were immunized with IFNγ 69-78 peptide using anti-CD40 and Poly(I:C) as adjuvant.6 days after immunization, splenocytes were stimulated with IFNγ 69-78 peptide for 6 hours as described in Methods and production of TNF by CD8 T cells was analyzed by flow cytometry.c.WT and Ifng -/-mice (on C57BL/6J background) were lethally irradiated and reconstituted with syngeneic WT or Ifng -/-BM cells depleted of T-and NK-cells.11 weeks after reconstitution, the resulting four groups of BM chimeras were immunized with IFNγ 69-78 and OVA 257-264 peptides using anti-CD40 and Poly(I:C) as adjuvant.6 days after immunization, splenocytes were stimulated with OVA 257-264 peptide for 6 hours as described in Methods and production of TNF by CD8 T cells was analyzed by flow cytometry (n = 3 for WT→WT, n = 4 for the other groups).Representative results of two independent experiments.Data are presented as the mean ± s.d. with NS: non-significant (P > 0.05) and *P < 0.05.Data were analyzed by two-tailed Student's t test (b) or one-way ANOVA with Holm-Sidak's multiple comparisons test (c).
-UEA1 + Ly51 -) from IL17A-GFP mice (n = 3 mice).d.WT and IL17A-GFP mice (on C57BL/6J background) were immunized s.c. with GFP 81-95 peptide in CFA or left unimmunized (WT only).14 days after immunization, I-A b GFP 81-95 tetramer-binding cells were magnetically enriched from pooled lymph nodes and spleens and quantified by flow cytometry.Representative flow cytometry plots gated on CD4 T cells (left) and absolute numbers of CD4 + CD44 + Tetramer + T cells (right) are shown.Representative results of two independent experiments (n = 4 mice per group).e.Comparison of the frequencies of GFP + hematopoietic cells in the thymi of IL17A-GFP mice and in IL17A-GFP→WT BM chimeras (analyzed ≥ 7 weeks after reconstitution) (n = 4 for IL17A-GFP mice, n = 6 for chimeras).Data are presented as the mean ± s.d. with NS: non-significant (P > 0.05).Data were analyzed by two-tailed Student's t test (d).